Sains Malaysiana 45(3)(2016): 451–458

Hydrogen Peroxide Induces Acute Injury and Up-regulates Inflammatory Gene Expression in Hepatocytes: An in vitro Model

(Hidrogen Peroksida Mengaruh Kecederaan Akut dan Mengatur Naik Ekspresi Gen Inflamasi dalam Hepatosit: Suatu Model in vitro)

LING LING LIAU¹, MAKPOL SUZANA², ABDUL GHANI NUR AZURAH³ & KIEN HUI CHUA¹*

¹Department of Physiology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan Yaacob Latif, 56000 Kuala Lumpur, Malaysia

²Department of Biochemistry, Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan Yaacob Latif, 56000 Kuala Lumpur, Malaysia

³Department of Obstetrics and Gynaecology, Faculty of Medicine, Universiti Kebangsaan Malaysia

Jalan Yaacob Latif, 56000 Kuala Lumpur, Malaysia

Received: 19 April 2015/Accepted: 28 September 2015

ABSTRACT

In the past, many in vitro hepatocyte injury models developed for liver regeneration used carbon tetrachloride as irritant chemical. Recently, carbon tetrachloride usage was prohibited due to serious deleterious effects to human and environment. There is an urgent need to develop a new acute chemical-induced hepatocyte injury model using other chemical compound to replace carbon tetrachloride. In this study, we used hydrogen peroxide (H₂O₂) to induced hepatocyte injury with HepG₂ as the liver cell model. HepG₂ injury was established by exposing the cells to CC₅₀ of H₂O₂ at the concentration of 2.4 mM, predetermined via MTT assay for 2 h exposure. Aspartate aminotransferase (AST) activity was measured to determine the extent of cellular injury and quantitative PCR was carried out to determine the expression of inflammatory genes of the cells 24 h after H₂O₂ exposure. The results showed that AST activity increased with time and peak at 24 h after H₂O₂ exposure. Quantitative PCR analysis demonstrated that expression of inflammatory genes (TGF-β1, MMP-3, NF-κβ, IL-8 and IL-6) increased significantly. In addition, the gene expression of GPX, an anti-oxidant enzyme was also increased significantly in response to oxidative stress. In summary, H₂O₂ demonstrated excellent capability in inducing oxidative injury to HepG₂ and together they represent an ideal acute chemical-induced injury model that can be used for liver regeneration study. Our results also provide input for inflammatory gene expression in the hepatocyte injury model.

Keywords: Hepatocytes; H₂O₂; inflammatory genes; in vitro; liver injury model

ABSTRAK

Banyak model kecederaan hepatosit in vitro yang telah dibangunkan untuk kajian regenerasi hepar pada masa lalu adalah berdasarkan kepada iritasi karbon tetraklorida. Kebelakangan ini, karbon tetraklorida telah dilarang penggunaaannya disebabkan ia boleh membawa kemudaratan kepada manusia dan persekitaran. Maka, satu model kecederaan hepatosit akut aruhan bahan kimia yang baru untuk menggantikan karbon tetraklorida kepada bahan kimia lain perlu dibangunkan dengan kadar segera. Dalam kajian ini, kami menggunakan hidrogen peroksida (H₂O₂) untuk mengaruh kecederaan hepatosit dengan menggunakan HepG₂ sebagai model sel hepar. Kecederaan HepG₂ diaruh dengan mendedahkan sel kepada CC₅₀ H₂O₂ pada kepekatan 2.4 mM yang telah dipratentukan melalui asai MTT selama 2 jam pendedahan. Aktiviti aspartat transaminase (AST) diukur untuk menentukan tahap kecederaan sel dan PCR kuantitatif dijalankan untuk menentukan ekspresi gen-gen inflamasi pada masa 24 jam selepas pendedahan kepada H₂O₂. Hasil kajian mendapati aktiviti AST meningkat dengan masa dan mencapai puncak kepekatan pada masa 24 jam selepas pendedahan kepada H₂O₂. PCR kuantitatif menunjukkan peningkatan ekspresi gen-gen inflammasi (TGF-β1, MMP_-3, NF-κβ, IL_-8 and IL_-6) secara signifikan. Selain itu, ekspresi gen GPX, sejenis enzim antioksidan juga meningkat secara signifikan sebagai respons kepada tekanan oksidatif. Secara kesimpulan, H₂O₂ menunjukkan keupayaan yang baik untuk mengaruh kecederaan oksidatif pada HepG₂ dan kombinasi H₂O₂ dengan HepG₂ menghasilkan model kecederaan hepatosit akut aruhan bahan kimia yang ideal untuk kajian regenerasi hepar. Di samping itu, hasil kajian ini juga memberi input terhadap ekspresi gen inflamasi dalam model kecederaan hepatosit.

Kata kunci: Gen inflamasi; hepatosit; H₂O₂; in vitro; model kecederaan hepatosit

REFERENCES

Alía, M., Ramos, S., Mateos, R., Bravo, L. & Goya, L. 2005. Response of the antioxidant defense system to tert-butyl hydroperoxide and hydrogen peroxide in a human hepatoma cell line (HepG₂). Journal of Biochemical and Molecular Toxicology 19(2): 119-128.

Bansal, M.B., Kovalovich, K., Gupta, R., Li, W., Agarwal, A., Radbill, B., Alvarez, C.E., Safadi, R., Fiel, M.I., Friedman, S.L. & Taub, R.A. 2005. Interleukin-6 protects hepatocytes from CCl4-mediated necrosis and apoptosis in mice by reducing MMP-2 expression. J. Hepatol. 42(4): 548-556.

Bernal, W., Auzinger, G., Dhawan, A. & Wendon, J. 2010. Acute liver failure. The Lancet 376(9736): 190-201.

Cesaratto, L., Vascotto, C., Calligaris, S. & Tell, G. 2004. The importance of redox state in liver damage. Ann. Hepatol. 3(3): 86-92.

Dong, W., Simeonova, P.P., Gallucci, R., Matheson, J., Flood, L., Wang, S., Hubbs, A. & Luster, M.I. 1998. Toxic metals stimulate inflammatory cytokines in hepatocytes through oxidative stress mechanisms. Toxicology and Applied Pharmacology 151(2): 359-366.

Galun, E. & Axelrod, J.H. 2002. The role of cytokines in liver failure and regeneration: Potential new molecular therapies. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 1592(3): 345-358.

Hartley, J.L. & Kelly, D.A. 2010. End stage liver failure. Paediatrics and Child Health 20(1): 30-35.

Herlong, H.F. 1994. Approach to the patient with abnormal liver enzymes. Hospital Practice 29(11): 32-38.

Hewitt, N. & Hewitt, P. 2004. Phase I and II enzyme characterization of two sources of HepG₂ cell lines. Xenobiotica 34(3): 243-256.

Hill, D., Marsano, L., Cohen, D., Allen, J., Shedlofsky, S. & McClain, C. 1992. Increased plasma interleukin-6 concentrations in alcoholic hepatitis. The Journal of Laboratory and Clinical Medicine 119(5): 547-552.

Jeong, W.I., Do, S.H., Jeong, D.H., Hong, I.H., Park, J.K., Ran, K.M., Yang, H.J., Yuan, D.W.,  Kim, S.B., Cha, M.S. & Jeong, K.S. 2006.  Kinetics of MMP-1 and MMP-3 produced by mast cells and macrophages in liver fibrogenesis of rat. Anticancer Research 26(5A): 3517-3526.

Kanebratt, K.P. & Andersson, T.B. 2008. Evaluation of HepaRG cells as an in vitro model for human drug metabolism studies. Drug Metabolism and Disposition 36(7): 1444-1452.

Knittel, T., Mehde, M., Grundmann, A., Saile, B., Scharf, J.G. & Ramadori, G. 2000. Expression of matrix metalloproteinases and their inhibitors during hepatic tissue repair in the rat. Histochemistry and Cell Biology 113(6): 443-453.

Li, G.P., Wu, L.F. & Pu, Z.J. 2008. Oxidative stress induces apoptosis in HepG₂ cells. Chinese Journal of Pathophysiology 24(1): 105-111.

Marion, M.J., Hantz, O. & Durantel, D. 2010. The HepaRG cell line: Biological properties and relevance as a tool for cell biology, drug metabolism, and virology studies. In Hepatocytes, edited by Maurel, P. New York: Springer. pp. 261-272.

McElwee, M.K., Song, M.O. & Freedman, J.H. 2009. Copper activation of NF-κB signaling in HepG₂ cells. Journal of Molecular Biology 393(5): 1013-1021.

Merrill, C.L., Ni, H., Yoon, L.W., Tirmenstein, M.A., Narayanan, P., Benavides, G.R., Easton, M., Creech, D.R., Hu, C.X. & McFarland, D.C. 2002. Etomoxir-induced oxidative stress in HepG₂ cells detected by differential gene expression is confirmed biochemically. Toxicological Sciences 68(1): 93-101.

Mitra, V. & Metcalf, J. 2009. Metabolic functions of the liver. Anaesthesia & Intensive Care Medicine 10(7): 334-335.

Okamura, M., Hashimoto, K., Shimada, J. & Sakagami, H. 2004. Apoptosis-inducing activity of cisplatin (CDDP) against human hepatoma and oral squamous cell carcinoma cell lines. Anticancer Research 24(2B): 655-662.

Oliveira, F.S., Silveira, T.R. & Matte, U. 2012. Relationship between serum transforming growth factor ß1 and liver collagen content in rats treated with carbon tetrachloride. Arquivos de Gastroenterologia 49(3): 232-234.

Poli, G. & Parola, M. 1997. Oxidative damage and fibrogenesis. Free Radical Biology and Medicine 22(1-2): 287-305.

Reeves, H.L. & Friedman, S.L. 2002. Activation of hepatic stellate cells-a key issue in liver fibrosis. Front. Biosci. 7(4): 808-826.

Sahreen, S., Khan, M.R. & Khan, R.A. 2011. Hepatoprotective effects of methanol extract of Carissa opaca leaves on CCl4- induced damage in rat. BMC Complementary and Alternative Medicine 11: 48.

Salazar-Montes, A., Delgado-Rizo, V. & Armendáriz-Borunda, J. 2000. Differential gene expression of pro-inflammatory and anti-inflammatory cytokines in acute and chronic liver injury. Hepatology Research 16(3): 181-194.

Selzner, M., Graf, R. & Clavien, P.A. 2003. IL-6: A magic potion for liver transplantation? Gastroenterology 125(1): 256-259.

Sun, Y., Tokushige, K., Isono, E., Yamauchi, K. & Obata, H. 1992. Elevated serum interleukin-6 levels in patients with acute hepatitis. Journal of Clinic Immunology 12(3): 197-200.

Weber, L.W.D., Boll, M. & Stampfl, A. 2003. Hepatotoxicity and mechanism of action of haloalkanes: Carbon tetrachloride as a toxicological model. CRC Critical Reviews in Toxicology 33(2): 105-136.

Wu, J., Danielsson, Å. & Zern, M. A. 1999. Toxicity of hepatotoxins: new insights into mechanisms and therapy. Expert Opinion on Investigational Drugs 8(5): 585-607.

*Corresponding author; email: ckienhui@hotmail.com